Regulation of the high-output pathway of nitric oxide synthesis involves proteins that bind to DNA sequences in the gene encoding inducible nitric oxide synthase (iNOS; NOS2) and proteins that bind to the enzyme itself. In the last award period we cloned the mouse iNOS promoter and initiated analysis of transcription factors participating in induction of iNOS by bacterial lipopolysaccharide (LPS) and interferon-gamma (IFNgamma). In addition, we characterized protein-protein interactions intrinsic to the iNOS tetramer--those necessary for formation of the apoprotein homodimer and for its attachment of calmodulin. The present grant seeks to: (i) clone and characterize novel transcription factors involved in the induction of the mouse iNOS gene by LPS; (ii) extend the analysis of transcriptional regulation to the human system; and (iii) clone and characterize proteins extrinsic to iNOS that are associated with the bipartite distribution of iNOS between cytosolic and vesicular compartments in macrophages. Specifically, we will further identify and clone LPS-activated transcription factors bound directly or indirectly to proximal promoter region D containing two LPS-responsive elements, NFkappaBd and the newly discovered LREAA, and analyze how LPS affects the expression, phosphorylation or subcellular localization of these factors. We have confirmed that the proximal human iNOS promoter responds to LPS stimulation when expressed in mouse cells and will identify the LPS-responsive promoter elements and transcription factors both in the murine context and upon expression in human cells responsive to physiologic complexes of LPS with CD14. We have identified a macrophage cell line in which a substantial portion of iNOS is associated with 50-80 nm vesicles as in primary macrophages, and we have detected polypeptides of approximately 121, 111, 101 and 43 kDa that co- immunoprecipitate with iNOS from a vesicle-rich fraction on density gradient ultracentrifugation. The yeast two-hybrid system or co- immunoprecipitation from this low-density fraction will provide routes to clone these proteins. We will characterize the interaction with iNOS in cell-free and cellular systems and assess their impact on macrophage antimicrobial and antitumor function.